Pharmaceutical Development and Technology

ISSN: 1083-7450 (Print) 1097-9867 (Online) Journal homepage: https://www.tandfonline.com/loi/iphd20

Imidazolium-based ionic liquid functionalized

mesoporous silica nanoparticles as a promising
nano-carrier: response surface strategy to
investigate and optimize loading and release
process for Lapatinib delivery

Parvaneh Peyvand, Zahra Vaezi, Mosslim Sedghi, Nima Dalir, Leila Ma'mani
& Hossein Naderi-Manesh

To cite this article: Parvaneh Peyvand, Zahra Vaezi, Mosslim Sedghi, Nima Dalir, Leila Ma'mani
& Hossein Naderi-Manesh (2020): Imidazolium-based ionic liquid functionalized mesoporous silica
nanoparticles as a promising nano-carrier: response surface strategy to investigate and optimize
loading and release process for Lapatinib delivery, Pharmaceutical Development and Technology

To link to this article: https://doi.org/10.1080/10837450.2020.1803909

View supplementary material

Accepted author version posted online: 04

Aug 2020.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=iphd20
Imidazolium-based ionic liquid functionalized mesoporous silica nanoparticles
as a promising nano-carrier: response surface strategy to investigate and
optimize loading and release process for Lapatinib delivery

Parvaneh Peyvanda†, Zahra Vaezia†, Mosslim Sedghia†, Nima Dalirb, Leila Ma'manic, and Hossein Naderi-Maneshd*

a
Department of Biophysics, Faculty of Biological Science, Tarbiat Modares University, PO Box: 14115-154,
Tehran, Iran.
b
Department of physical chemistry, Faculty of Basic Science, Tarbiat Modares University, PO Box: 14115-154,

t
Tehran, Iran.

ip
c
Department of Nanotechnology, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural
Research, Education and Extension Organization (AREEO), Karaj, Iran.
d
Department of Biophysics / Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, PO

cr
Box: 14115-154, Tehran, Iran.

us
*
Corresponding author: Tel./Fax: +98-21-82884410; naderman@modares.ac.ir (Hossein Naderi-Manesh)
†
P. Peyvand, Z. Vaezi, and M. Sedghi contributed equally as first authors.

Abstract
an
Imidazolium-based ionic liquid functionalized PEGylated mesoporous silica nanoparticles
MCM-41 (denoted as [ImIL-PEGylated@MCM-41] NPs) is synthesized and evaluated as an
M
efficient and reliable pH-sensitive nano-carrier for controlled release of cationic Lapatinib (Lap)
drug. This nano-DDS was fully characterized by dynamic light scattering, scanning electron
microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, N2 adsorption–
d

desorption measurement, and differential scanning calorimeter. Furthermore, the drug loading
e

content and in-vitro drug release profile were studied. The entrapment and loading efficiency of
the optimized formulation for Lap were 91 ± 2.0% and 32.21 ± 2.70%, respectively. The results
pt

of cytotoxicity assay demonstrated that ImIL-PEG@MCM-41 has no significant toxicity on both

cancerous and normal cell lines and the anticancer activity of Lap@ImIL-PEG@MCM-41 was
ce

comparable to free drug in case of human breast cells (SKBR3) and human embryonic kidney
293 cells (HEK-293). Meanwhile, three-dimensional (3D) cell culture was performed by
Ac

multicellular tumor spheroids for understanding of cell response to drugs in physiologically 3D

microenvironments. The results of Lap@ImIL-PEG@MCM-41 uptake during 48 hours showed a
gradual release of the Lap through the multicellular tumor spheroids. This showed that the pH-
responsive controlled release of Lapatinib leads to the satisfactory results in the in vitro breast
cancer therapy.

Keywords: Mesoporous silica nanoparticles, Ionic liquid, Lapatinib, pH-sensitive drug delivery,
Controlled release, Multicellular tumor spheroids, Breast cancers.
1. Introduction
Encapsulation of drugs within biocompatible and nontoxic nanoparticles for controlled and
sustained release to the target is one of the strategies for cancer therapy (Mohammadi et al. 2019;
Rezaei et al. 2020). The controlled nano-drug delivery systems (nano-DDS) not only is able to
prevent the side effects of the conventional dosage such as potential degradation and toxic effects
of drugs, but it can also improve the drug release performance, bioavailability and stability by
modifying the surface physicochemical properties of nano-DDS (Luchini et al. 2018; Mehrdad-
Vahdati et al. 2019). Most of the chemical drugs, from a therapeutic point of view, have high
levels of toxicity and the potential of causing various side effects (Liang et al. 2010; Zahra Vaezi
2020). Lapatinib (Lap) is one of the hydrophobic drugs, which composed of two members of the

t
ip
epidermal growth factor receptor family (EGFR family) HER1 and HER2, acts as tyrosine kinase
inhibitor (Zhang D et al. 2008; Escudero-Ortiz et al. 2013). In some breast cancers, over

cr
expression of HER1 and HER2 has been reported (Roy and Perez 2009) and for the treatment of
HER2-positive metastatic, Lapatinib was approved by federal drug administration (FDA) (Petrov

us
et al. 2006; Zhang D et al. 2008). Since more than 99% of the free drug binds to alpha-1 acid
glycoprotein and albumin, it is necessary to protect Lapatinib from plasma protein adsorption by
encapsulating it inside a carrier in the blood circulation system until eventually get to its target
sites (Koch et al. 2009).
an
One of the advantages of drugs loaded in the NPs is their accumulation and release in tumors,
while free drugs or small molecules rapidly subjected to renal filtration, therefore, the retention
M
time of the packed drugs in NPs is ten times higher than free drugs at the site of the tumor
(Daglar et al. 2014; Zhang X et al. 2014). Mesoporous silica nanoparticles (MSNs) can act as
effective nanocarriers and a promising candidate in the field of drug delivery (Maleki and
d

Hamidi 2016; Esfandyari et al. 2020). MSNs with extensive surface areas are used to deliver
e

large doses of drug in a controlled manner because they are stable over a wide range of
temperature and pH (Faraji and Wipf 2009; Muhammad et al. 2014). By controlling the size,
pt

surface chemistry, shape, and hollow structure of them, they are superior to other nano-drug
delivery systems (DDSs), especially in properties such as biocompatibility and excretion from
ce

the body in the treatment of cancer (Tang et al. 2012; Chang F-P et al. 2013; Hashemi et al.
2020). Furthermore, MSNs have been studied as bioavailability enhancing systems, e.g., many
Ac

hydrophobic drugs like Valsartan (Biswas 2017), curcumin (Hartono et al. 2016),and telmisartan
(Zhang Y et al. 2012) have successfully been encapsulated into MSNs and have proven to give a
remarkable enhancement in bioavailability. This methodology has been used in the formulation
of Lapatinib-encapsulated MSNs to improve certain free drug delivery shortcomings such as
poor stability, insolubility in water, side effects and low selectivity to be surmounted. Moreover,
it is required to synthesize nano-DDS with pH-responsive property to enhance therapeutic
application of them. The pH-sensitive nano-DDS are smart and applicable in cancer therapy
because the pH of tumor microenvironment is slightly more acidic than the pH of normal tissues,
thereby tumor tissues has more advantage to absorb this type of nano-DDS (Muhammad et al.
2011; Mei et al. 2012). It is notable that, the proficiency of MSNs is beholden to the high pore
volume with the uniform distribution, silanol functionalization opportunity, specific surface area,
and the hydrothermal stability (Ma'mani et al. 2014; Xia et al. 2018; Sakhtianchi et al. 2019).
Apart from these properties, the convenient surface modification and functionalization of MSNs
improved their efficiency and intelligence as nano-DDS for control release of embedded drug
(Hosseini et al. 2011; Li et al. 2019). The PEGylation of nanoparticles has been commonly used
as an effective approach to decrease the non-specific binding of nanoDDSs to blood proteins and
also to enhance the permeability and colloidal stability of nano-DDSs in aqueous medium (He et
al. 2010). The introduction of efficient and low-cost anticancer DDSs will be of great importance
for the integration of three-dimensional cubic Ia3d MSNs with various surface characteristics.
In this study, the imidazolium-based ionic liquid functionalized PEGylated MCM-41 ([ImIL-

t
PEG@MCM-41]) is synthesized as a pH-responsive nanocarrier for Lap delivery, improving the

ip
absorption and bioavailability of Lap in the cancerous cells. Furthermore, cytotoxicity and
release behavior of drug-loaded, specially, the effect of tumor microenvironment and pH on drug

cr
release were studied. It was found that this biocompatible nano-DDS is a good candidate for the
breast cancer treatment and a robust tool in biomedicine usage.

us
2. Materials and Methods

2.1. Materials and cell culture

an
Tetraethylorthosilicate (TEOS), hexadecyltrimethylammonium bromide (CTAB), sodium
M
hydroxid, toluene, 3-(aminopropyl) trimethoxysilane (APTMS), 3-chloropropyltriethoxysilane
(CPTMS), N-methylimidazole, and polyethylene glycol (PEG3000) were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Lapatinib ditosylate (Tykerb, Tyverb, and GW-572016) was
d

prepared from BioVision, Inc. (925.46MW). Acetonitrile was from Merck (Germany) in
analytical grade. DMEM (high glucose) culture medium was purchased from sigma (USA).
e

Dulbecco's phosphate buffered saline (D8662), fetal bovine serum (FBS, F2442) and MTT salt
pt

([3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]) (M5655) were from Sigma.

HER2+ human breast cancer (SKBR3) and HEK-293 (HER2-) cells were grown as a monolayer
ce

in DMEM (high glucose) medium supplemented with 20% FBS and containing 1% (v/v)
penicillin-streptomycin (10,000 U.mL-1) at 37 °C and 5% CO2. For in vitro screening, the cells
were dispersed in appropriate plates at exponential phase of growth.
Ac

2.2. Apparatus
The mean diameter and zeta potential of NPs were determined by dynamics light scattering
(DLS) measurements using Malvern Zeta-sizer ZS series and scattering particle size analyzer
(Malvern Co, UK). To investigate the evaluation of size as well as the morphological features of
nanoparticles, scanning electron microscopy (SEM) was applied. For this purpose, NPs samples
were covered with gold in vacuum, and then examined by a FE-SEM (Hitachi S-4160). FT-IR
spectra were recorded by Nicolet IR100 FT-IR spectrometer to determine the interaction of
polymers, also, polymers and drug. Thermo-gravimetric analysis (TGA) (Perkin Elmer TGA-7),
X-ray diffraction (XRD) measurement was performed using a Philips X'pert MPD model with
Co tube, λ=4.7986 Aͦ, step size 0.04 s-1, current 30 mA, and V=40KV and nitrogen adsorption
isotherms were measured by a volumetric gas adsorption apparatus (BEL Japan, Belsorp-max) at
77 K.
2.3. Preparation of nanocarrier
2.3.1. Synthesis of MCM-41
CTAB (1.02 g, 2.8 mmol) was initially dissolved in NaOH (2 M) and the mixture was vigorously
stirred at 80 ˚C under vigorous stirring, adding drop-wise TEOS (5.0 mL) to the reaction mixture.
The temperature was maintained at 80 ˚C for 2 h, and then white precipitate was produced and
isolated by filtration (0.45µm), washing several times with water and EtOH then dried under

t
ip
vacuum oven. The surfactant was removed via calcination at 540 ºC for 6 h to obtain the fine
MCM-41 (Chen et al. 2015).

cr
2.3.2. Synthesis of MCM-41-NH2
While the MCM-41 (100 mg) was dispersed in toluene, APTMS (1.0 mmol) added step by step,

us
then refluxed for 24 h. The resulted material was filtered, washed with toluene and methanol
then dried under vacuum (Yoncheva et al. 2014; Chen et al. 2015).
2.3.3. Synthesis of PEGylated MCM-41(PEG@MCM-41)
an
PEGylated MCM-41 (PEG@MCM-41) was synthesized with reaction of MCM-41-NH2 and
PEG3000-OTs (MW=3000) (Ma'mani et al. 2014). 100 mg MCM-41 and 0.5 mmol PEG3000-OTs
M
was mixed in 100 mL dry MeOH, and this mixture was incubated 12 h in room temperature
(R.T.). Then later solution was centrifuged, and finally the precipitate was washed several times
with deionized water (DW) and EtOH and dried under vacuum.
d

2.3.4. Synthesis of ImIL-PEG@MCM-41

e

Firstly, CPTMS (1g, 5 mmol) was added to a suspension of as-synthesized PEGylated MCM-
pt

41(PEG@MCM-41, 1g) in 30 mL toluene. The suspension was refluxed for 72 h. The solid was
filtered, exhaustively washed and dried under vacuum to obtain chloropropyl functionalized
ce

PEGylated MCM-41(Fallah-Bagheri et al. 2012). Chloropropyl functionalized PEG@MCM-41

(1g) was dissolved in 60 mL toluene, N-methyl imidazole (5 mmol) was slowly added, and then
the mixture was refluxed for 24 h. To obtain ImIL-PEG@MCM-41, the resulted precipitate
Ac

washed with diethyl ether and water and dried under vacuum at 40 ˚C for 24 h.
2.3.5. Synthesis of FITC labeled ImIL-PEG@MCM-41 (FITC@ImIL-PEG@MCM-41)
APTES (5 mL) and fluorescein isothiocyanate (FITC) (1 mg) were stirred in dry EtOH (3 mL) at
R.T. for 24 h in the dark; then a suspension of ImIL-PEG@MCM-41 (200 mg) in 7 mL EtOH
was added to the first solution and the whole mixture stirred for 48 h in the dark. For obtain
FITC@ImIL-PEG@MCM-41, the precipitate washed with diethyl ether and water then dried
under vacuum at 40 ˚C for 24 h.
2.4. Drug loading and release
2.4.1. Loading and entrapment efficiency of drug by using HPLC method
To determine the entrapment efficiency of Lapatinib, a certain volume of Lap@ImIL-
PEG@MCM-41 was lyophilized and re-dispersed in a defined volume of DMSO; the
supernatant was collected after centrifuging at 20,000 rpm for 20 min. The chromatographic
analysis was performed on reverse-phase HPLC (AKTA purifier) instrument consisting of binary
pump system and equipped with C18 column (4.6 × 50 mm, 5.0 μm) and UV detector. Injection
volume was 20μl and all samples were measured at 268 nm. The mobile phase was composed of
double distilled water and acetonitrile in a gradient of 70:30 to 50:50 (v/v) with a flow rate of 1
mL.min-1. Data acquisition and quantification were performed on Clarity software. The amount
of Lapatinib was calculated according to a calibration curve (standard curve of Lap in DMSO)

t
ip
and then the following equations were used to determined efficiency of entrapment and loading:

cr
a m o u n t o f d r u g in N P s
E n tr a p m e n t e ffic ie n c y (% )   100
in itia l a m o u n t o f d r u g

us
a m o u n t o f d r u g in N P s
L o a d in g e ffic ie n c y (% )   100
w e ig h t o f N P s

2.4.2. Drug release study an

To verify the feasibility of using this kind of ImIL-PEG@MCM-41 for intracellular drug
delivery in future cancer chemotherapy, in vitro release behaviors has been performed under
different pH values of 5.0 and 7.4, which imitated local tumor microenvironment and normal cell
M
environment, respectively (Anitha et al. 2011). The investigation of drug release for a period of
48 h was performed as described in previous study (Bisht et al. 2007). A certain amount of
d

Lap@ImIL-PEG@MCM-41 was dispersed in 30 mL phosphate buffer (pH 5.0), then the

solution was divided into 30 vials and incubated in water bath shaker at 37 °C (Richeldi et al.
e

2014). Then three vials were taken out at definite time, centrifuged at 2000 rpm for 2 min to
pt

discard supernatants containing drug loaded nanoparticles then the pellets of free drug were
dissolved in a certain amount of DMSO. The released drug was measured by reverse-phase
ce

HPLC and this study was also repeated for neutral pH with PBS (pH 7.4).

2.5. Cytotoxicity study

Ac

To evaluate cytotoxicity of Lap@ImIL-PEG@MCM-41 as well as MSN and free Lap, MTT

assays were performed. In 96-well plates, the cells (SKBR3 and HEK-293) were seeded at a
density of 1×104 cells per well and incubated for 48 h. After this time, the medium was removed
and refilled with new medium containing the sample in a serial concentration pattern. All
treatments were examined in triplicate and the amount of living cells was determined by addition
of the fresh medium containing 0.5 mg.mL-1 of MTT salt to the wells. After 3–4 h, the medium
was removed then formazan crystals were dissolved in 200 μL of DMSO, and the cell viability
was directly correlated by the background subtraction at 630 nm from the solution absorbance at
550 nm using microplate readers (MQuant Biotech U.S.).
 A te s t
c e ll v ia b ility (% )   100
A c o n tr o l

Where the Atest and Acontrol were obtained by subtracting the OD630 from the OD570 and the IC50
values were calculated by nonlinear regression analysis using the equation for sigmoid plot in
GraphPad Prism 8.3.0.

2.6. In vitro tumor study

To scrutinize how this system release Lapatinib through the tumor, a three-dimensional culture
of SKBR3 cells was conducted, that is, using non-adherent agarose made microwells, SKBR3
cells were cultured and formed spheroids. The cells with density of 5 × 105 cells were loaded on

t
the microwells and standard cell culture conditions were applied, then after 4-5 days of cell

ip
loading and after the spheroids were grown enough to be treated, each group of spheroids was
exposed to 5µg.mL-1 of Lap@ImIL-PEG@MCM-41, then 12 hours after treatment of the in-vitro

cr
tumors, the microwells was rinsed three times with PBS for 30 min intervals, then the fluorescent
images were gathered every 12 hours until 48 hours.

us
2.7. Statistical analysis
an
To analyze the results of cytotoxicity assay, multiple comparisons analysis was conducted using
GraphPad Prism 8.3.0. The outcome is presented in the results section.
M
3. Results and discussion

3.1. Preparation and characterization of MCM-41

d

The functionalized mesoporous silica nanoparticles by imidazolium ionic liquid, were prepared
e

via grafting of organic spacers on the surface of MSNs in two steps. In the synthetic procedure,
the organic soft templating agent (CTAB) was applied for providing MSN based nanocarrier.
pt

The surface of MCM-41 NPs was PEGylated to enhance the permeability and colloidal stability
in an aqueous dispersion. Then for improving the controlled loading and release of cargo (Lap),
ce

imidazolium based ionic liquid was covalently grafted on the surface of PEG@MCM-41. A
schematic of the synthesis of imidazolium based ionic liquid functionalized mesoporous silica
nanoparticles (ImIL-PEG@MCM-41) is presented in Scheme 1, and also its role as a nano-
Ac

carrier for the delivery of Lap to cancerous cell. All the synthesized samples were characterized
by using FT-IR, SEM, TGA, BET, DLS and XRD techniques. Next, the ability of this porous
DDS was investigated for in-vitro delivery of Lap into SKBR3 and HEK-293 cell lines.

Scheme 1.

The morphology and meso-structured of MCM-41 were studied by XRD and SEM. Figure 1a
shows the XRD patterns for MCM-41 silica NPs in a range from 2θ (2°-10°), with three peaks
corresponding for the planes (100), (110) and (200) located at 2.3, 4.1 and 4.7° of 2θ,
respectively. The characteristic peak (100) is more intense and sharp, while, the peaks of the
planes (110) and (200) are more defined, attributing to the 2D hexagonal structure of MCM-41
silica (Meléndez-Ortiz et al. 2013). As shown in Figure 1b, the SEM image revealed short rod-
like morphology with a smooth surface for MCM-41 silica NPs and the average size of
approximately 146 nm. Furthermore, the SEM images illustrated an average size of 201.5±9.4
nm, with a narrow particle size distribution (PDI=0.05), Figure 1c, verified the DLS results (Qi
et al. 2011).

Fig. 1.

The zeta potential of the modified carrier shifted from negative to positive side, demonstrated a

t
ip
successful grafting of amine group on the external surface. The negatively charge of MCM-41 (-
22.9 mV) change to positive charged (3.3 mV) after PEGylation and show a further shift to the

cr
more positive value (+17.2 mV) due to the presence of N-methyl imidazole ionic liquid and Lap
in the nanoparticles of Lap@ImIL-PEG@MCM-41, which arises from the fully ionization of the

us
ionic liquid groups. This increment in the zeta potential value of bare and functionalized MCM-
41 engulfed nanoparticle could be attributed to the positive zeta potential of Lap drug.
The FT-IR spectra of MCM-41, PEG@MCM-41, ImIL-PEG@MCM-41, Lap and Lap@ImIL-
an
PEG@MCM-41 are shown in Figure 2 and Figure S1, supplementary material (ESM). The
amine functionalized MCM-41was confirmed by two broad bands at 3425 and 1628 cm-1, which
related to the N-H stretching and bending vibrations, respectively (Yoncheva et al. 2014). The
M
presence of amine groups in the structure of the mesoporous nanoparticles was also confirmed by
energy dispersive X-ray fluorescence (EDX) analysis and the results were shown in Figure S2,
ESM. New peaks at 1232, 1666 and 2941 cm-1 confirmed the C-H vibrations of the aliphatic
d

chain and the PEGylation of ImIL-PEG@MCM-41 shows a strong OH peak at 3500 cm-1 and C-
e

H vibrations of polymer chain at 2925 cm-1. The presence of N-methyl imidazole ionic liquid
was verified by new peaks in 1600 cm-1 and 1000-500 cm-1 region to C=C and C-H vibrations in
pt

aromatic ring of ionic liquid (Chen et al. 2015). An alteration in the C-H bending at 900–600
cm−1 expressed the aromatic rings of Lapatinib (Meta positions) in the spectrum of Lap@ImIL-
ce

PEG@MCM-41. Moreover, the changes in the N-H vibrations were observed in aromatic
compounds, overlapping the aromatic C-C ring absorptions (in pairs at 1600 cm−1 and 1475
Ac

cm−1) (Mobasseri et al. 2017). The favorable output of amine grafting with drug was disappeared
from the N-H bending peak and C-H stretching peak at 1585 cm-1 and 2915 cm-1 ,respectively, in
the Lap@ImIL-PEG@MCM-41 spectra (Ebrahimi-Gatkash et al. 2017). This indicated that the
ImIL-PEG@MCM-41 can interact with Lap to specifically be able to trap drug and help in the
controlled drug release.
Fig. 2.

The N2 adsorption/desorption isotherm, Brunauer-Emmett-Teller (BET) and Barrett-Joyner-

Halenda (BJH) were exploded to confirm the porosity and meso-characteristic of the nanocarrier.
Therefore, the specific surface area and pore diameter and size distribution of synthesized
mesoporous nanocarrier were determined by BET and BJH analyses. As shown in Figure 3, the
adsorption-desorption isotherms can evaluated as type IV (Zeng et al. 2012). Our analysis
showed that after functionalization and loading process, the amount of the adsorbed N2 decreased
but their hysteresis loop had an identical pattern.
Fig. 3.

The specific surface area, pore volume, and the pore size of MCM-41, ImIL-PEG@MCM-41,
and Lap@ImIL-PEG@MCM-41 are present in Table 1. The pore parameters of MCM-41
significantly decrease after surface modification and drug loading which confirmed successfully
grafting and loading of Lap into the pores of nanocarrier. The surface area (689.8 m2.g-1) and

t
pore volume (0.79 cm3.g-1) was determined by BET and the pore diameter evaluated about 3.62

ip
nm by the BJH method (Yoncheva et al. 2014). After surface functionalization and drug loading,
the BET surface area decreased to 471.2 and 362.1 (m2.g-1), respectively. As well as, the pore

cr
size changed from 3.01 nm to 2.50 nm during the drug loading into the surface of ImIL-
PEG@MCM-41. The gradual decrease in surface areas and pore sizes demonstrated that the

us
covalently functionalization of many functional groups were successfully accomplished on the
surface and into the pore of the MCM-41. Furthermore, the successful loading of Lap in ImIL-
an
PEG@MCM-41 was indicated by the secondary decrease.

Table 1
M
TGA thermo-gram of MCM-41, ImIL-PEG@MCM-41 and Lap@ImIL-PEG@MCM-41 NPs is
depicted in Figure 4. This figure shows a weight loss from 25 to 900 °C in N2 atmosphere
d

attributed to the decomposition of the organic moieties, then the loading of Lap and grafting of
e

organic functions on the surface of NPs was evaluated. As shown in Figure 4, the unmodified
MCM-41 showed two distinct regions of weight loss. The first region of mass loss (3.2%) occurs
pt

in temperature range between 25 and ~100 °C, which can be attributed to the thermo desorption
of physically adsorbed water molecules. The second region, in the temperature range from 100 to
ce

900 °C, the weight loss is almost negligible (1.0%) and corresponds to the silanol groups
condensation (Kim et al. 2009; de Oliveira Freitas et al. 2017), whereas the weight loss of ImIL-
PEG@MCM-41 was 30.10% in the same temperature. In other words, the total amount of PEG
Ac

and IL moieties in ImIL-PEG@MCM-41 is around 28% (w/w) (Abdous et al. 2017; Shah and
Rajput 2019). The larger the percentage of mass loss, the higher the degree of functionalization
of MCM-41(Jaroniec et al. 1997). Also, the weight loss of Lap@ImIL-PEG@MCM-41 was
58%, which it can be concluded that 30% Lap was loaded in ImIL-PEG@MCM-41.
Fig. 4.

3.2. Entrapment and loading efficiency

Two imperative parameters for drug delivery carriers are entrapment efficiency and loading
efficiency. The higher entrapment and loading efficiency will allow the more availability of drug
around the target, thereby, the required medication dose for treatment reduced with the reduction
of systematic side effects. As shown in Figure 5, both entrapment/loading efficiency for Lap
were assessed in different weight ratio of drug to nanoparticles. In Figure 5a, when the weight
ratio of Lap to NPs was <0.5, the entrapment efficiency increased with an increase in the ratio of
Lapatinib to ImIL-PEG@MCM-41, while the meaningful changes were not observed in the
entrapment efficiency at the ratio >0.5. Whereas, the loading efficiency revealed an increase in
pattern by increasing the Lap/NPs ratio (Figure 5b). The highest entrapment efficiency 91% and
the maximum loading efficiency 33% were obtained at 0.5 (w/w) ratio of Lap/ImIL-
PEG@MCM-41. These results for Lap in ImIL-PEG@MCM-41 indicated a strong intermixing
of Lap with IL can increase the solubility and entrapment efficiency of Lap as a hydrophobic

t
drug. Ravar et. al. have demonstrated that Lap encapsulated into liposome around 52% and

ip
Dextran sulfate-chitosan nanoparticles were able to entrap 76.7% of this drug which reported by
Mobasseri et. al. (Ravar et al. 2016; Mobasseri et al. 2017). Compared with their results, the

cr
obtained entrapment efficiency in this study is acceptable.

us
Fig. 5.

3.3. Drug release pattern an

The local medium of the tumor cells are acidic due to highly lactic acid production and
accumulation of end products in their environment (De Milito and Fais 2005). Recently, drug
release pattern of pH-sensitive MSNs-carrier was investigated by applying powerful statistical
M
central composite design combined with response surface methodology (CCD-RSM). The results
showed that the drug release at acidic pH is more than basic pH, which verifies its pH-responsive
d

property and the maximum drug release was obtained at 5.16 of pH (Abdous et al. 2017). As
shown in Figure 6, in vitro drug release pattern was obtained at pH 5.0 and 7.4, representing
e

acidic and neutral medium, respectively. For a period of 48 h, approximately 73% of Lap was
pt

released under acidic condition while the release was less than 54% in pH 7.4 (Hobbs et al.
1998). To improve the controlled release of Lap, imidazolium based ionic liquid was covalently
ce

grafted on the surface of the PEGylated MCM-41, which is composed of NH functional groups
for synthesizing pH-responsive nanocarriers. At neutral medium, the functionalized mesoporous
silica could accommodate the drug molecules because the mesoporous were capped by the
Ac

gatekeepers, while at acid intercellular environment, the gatekeeper would be removed to release
the loaded drug due to the hydrolysis of imidazolium ionic liquids (Chen et al. 2015). The total
amount of release during 48 h divided into two phases: (1) distribution of drug due to rupture
within the first 4 h and (2) the controlled and slower release of Lap between the 5th and the 48th
hour. The drug located close to or adsorbed on NPs’ surface probably induces the initial release
while the later sustained release pattern was depended to drug into the pores of ImIL-
PEG@MCM-41. Additionally, the facilitated release of encapsulated drug and increase of IL
turgidity caused by the protonation of the amine groups of IL in acidic pH, which these results
agree with the findings of other studies (Chang D et al. 2016). The prolonged release of drugs
with short half-life was important for maintaining a therapeutic dose over a long period of time
(Siegel and Rathbone 2012). It was reported that the loading of Lap into liposome resulted in
initial release of >60% in 4 h and a sustained release over 40 h (Grimaldi et al. 2016).

Fig. 6.

The release profile of lapatinib from different nano-drug carriers were compared in controlled-
release formulations at pH= 5.0 and 7.4 (Table S1). As shown in Table S1, the release profile of
free lapatinib exceeded 80%, after 12 h, indicating that the dialysis bag had minimal rate-limiting
effect on lapatinib release. However, the lapatinib release from our nano-carriers was
characterized by a comparable controlled pattern with other systems; also, entrapment and
loading efficiency have improved compared to the previous studies (Mobasseri et al. 2017; Li et

t
al. 2019).

ip
3.4. In vitro cytotoxicity studies

cr
MTT assay was applied for evaluating the in vitro toxicity of Lap@ImIL-PEG@MCM-41 NPs
in the case of HER2 positive (SKBR3) and HEK-293 cells in different incubation time (Figure

us
7A) and the IC50 values were calculated by nonlinear regression analysis using the equation for
sigmoid plot in Figure 7B. Furthermore, the effects of free ImIL-PEG@MCM-41 and Lap on
both cells were assayed as negative and positive control, respectively. The experiments were
an
separately performed in triplicate for the SKBR3 cancer cell line (Figure 7A a) as well as HEK-
293 normal cell lines (Figure 7A b) in 24 h and 48 h incubation time. In Figure 7A a,
M
Lap@ImIL-PEG@MCM-41 showed satisfactory anti-cancer effect on SKBR3 cells in the 24 h
exposure with IC50 of 8.5 μg.ml-1, even though it was relatively worse in comparison to free drug
with IC50 of 4.7 μg.ml-1. The anticancer activity of Lap@ImIL-PEG@MCM-41 on SKBR3 cells
d

was found to be very close to the free drug in the 48th hour with IC50 of 4.6 μg.ml-1 and 4.8
μg.ml-1 for free drug and Lap@ImIL-PEG@MCM-41, respectively. After 48h, cell viability
e

decreased at the SKBR3 cells that treated with Lap@ImIL-PEG@MCM-41 in comparison with
pt

HEK-293 cells at all concentrations of the experiment. This is due to the controlled release of the
drug from nano-DDs and overexpression of Lapatinib receptors. Moreover, the IC50 values
ce

indicate that the toxicity effect of drug on both treated cell lines increased at 48h in comparison
with the cells treated for 24h. As shown in Figure 7A b, the toxicity effects of free drug and
nano-DDs loaded drug on normal cells were similar to cancer cells, although their toxicity on
Ac

cancer cells were less due to the lack of overexpression of Lapatinib receptors. Lap@ImIL-
PEG@MCM-41 NPs suppresses the proliferation of SKBR3 cancer cell line in comparison with
the HEK293 normal cell line. According to IC50 values of 48h treatment (Figure 7B), we choose
maximum concentration of (5 µg.ml-1), because this concentration show the greatest toxicity on
cancer cells and the less effect on normal cells. Moreover, ImIL-PEG@MCM-41 NPs did not
show any side effects on the cell lines even at high concentrations.
Fig. 7.

The statistical analysis was performed for cytotoxicity assay of SKBR3 cells in 24 and 48h
treatments, Sidak's multiple comparisons test for analyzing between cell line responses to
different treatments and Tukey’s multiple comparison tests for comparing the cell response to
different treated formulations, see ESM for more details. The results demonstrated that ImIL-
PEG@MCM-41 has no significant toxicity on both cancerous and normal cell lines and
Lap@ImIL-PEG@MCM-41 had satisfactory anticancer activity. The protection of Lapainib
against plasma proteins and early release from blood circulation is an important target for the
delivery of anti-cancer drugs (Koch et al. 2009). In this study, the ImIL-PEG@MCM-41 acted as
Lap carrier with the ability to protect its activity and showed that nano-DDS are biocompatible
and can be used as drug delivery among intracellular experiments.

3.5. Cellular uptake study

t
Biocompatibility is an important factor for anticancer drug delivery and the accuracy of that was

ip
performed by MTT assays. Cellular uptake of ImIL-PEG@MCM-41 was studied by fluorescent
imaging at different times, whereas; ImIL-PEG@MCM-41 labeled with FITC and cell nucleus

cr
stained with Hoechst 33342 dye. FITC can monitor the pathway of treatment process and its
distribution was all over the cytoplasm (green florescence) except for nuclei (blue florescence).

us
The in vitro internalization of nano-DDS at the 2st and 6th hour is shown in Figure 8 (e, f) and
Figure S3. It was observed that the rate of internalization of nano-DDS increased with exposure
time. As shown in Figure 8, Lap@ImIL-PEG@MCM-41 (8d) displayed the more cytotoxicity
an
for cancer cells than free Lap (8b) due to the cleavage of ionic liquid and drug bonds under the
cancerous microenvironment, promoting Lap effectively release from Lap@ImIL-PEG@MCM-
M
41 and performing a great tumor inhibition result. Also, the morphology of cancer cells became
spherical after incubation with Lap@ImIL-PEG@MCM-41 compared to the blank ImIL-
PEG@MCM-41 (8c) and control (8a). In addition, the less cell availabilities observed in the
d

higher concentrations of Lap@ImIL-PEG@MCM-41. These results were explained by the acid

intercellular environment trigged the removal of the gatekeeper. By this controllable on-off of
e

the mesoporous silica nanoparticles, the release of the loaded drug would be controlled, which
pt

effectively enhanced the tumor inhibition effects.

Fig. 8.
ce

3.6. Tumor uptake study

Three-dimensional (3D) cell culture using multicellular tumor spheroids (MCTS) can bridge the
Ac

gap between in vitro monolayer cell culture assays and in vivo assays and bring us a better
understanding of cell response to drugs in physiologically 3D microenvironments (Galateanu et
al. 2016; Yu et al. 2019). In this study, the results of MCTS uptake of the Lap@ImIL-
PEG@MCM-41 during 48 hours showed a gradual release of the Lap@nano-DDS through the
MCTSs. Furthermore, the comparison between control spheroids and the treated spheroids
showed a slight decrease in the size of the spheroids.
Fig. 9.

These results strongly indicated that the acidic protons effectively cleaved the electrostatic bonds
between Lapatinib and the methylimidazolium head group attached onto the surface of MSN. As
a result, the pH values of the medium has pivotal role in the release rate of Lap from ImIL-
PEG@MCM-41.

4. Conclusion
Drug delivery system of mesoporous silica nanoparticles modified with PEG and N-methyl
imidazole ionic liquid was prepared successfully for controlled release of cationic aromatic drug
Lapatinib. This IL coating was partially detached from the surface of MSNs in the acidic
condition, thus, the coating blocked the pores as ‘‘gatekeepers” for drug controlled release.
ImIL-PEG@MCM-41 had the size around 150 nm and high drug loading content. An entrapment
efficiency of 91% was observed and controlled drug release within duration of 48 h. In vitro

t
release of drug indicated that Lap@ImIL-PEG@MCM-41 was very sensitive to pH, with

ip
increasing acidity, its release rate increases. It is notable that the amount of Lap released from
Lap@ImIL-PEG@MCM-41 can be efficiently controlled by the acidity of medium. This study

cr
demonstrated that ImIL-PEG@MCM-41 nanoparticle with negative zeta potential, hydrophilic
nature and the non-covalent interaction between ionic liquid and drug has the potential to act as

us
an efficient delivery system for the encapsulation and controlled release of hydrophobic drugs.

Acknowledgment an
The authors would like to kindly acknowledge all the supports and funding from Tarbiat
Modares University (research grant No IG-39708).
M
Appendix A. Supplementary data
d

Conflict of interest
e

The authors declare that they have no conflicts of interest.

pt
ce
Ac
Reference

Abdous B, Sajjadi SM, Ma’mani L. 2017. β-Cyclodextrin modified mesoporous silica nanoparticles as a
nano-carrier: response surface methodology to investigate and optimize loading and release processes
for curcumin delivery. Journal of Applied Biomedicine. 15(3):210-218.
Anitha A, Deepagan V, Rani VD, Menon D, Nair S, Jayakumar R. 2011. Preparation, characterization, in
vitro drug release and biological studies of curcumin loaded dextran sulphate–chitosan nanoparticles.
Carbohydrate Polymers. 84(3):1158-1164.
Bisht S, Feldmann G, Soni S, Ravi R, Karikar C, Maitra A, Maitra A. 2007. Polymeric nanoparticle-
encapsulated curcumin (" nanocurcumin"): a novel strategy for human cancer therapy. Journal of
nanobiotechnology. 5(1):3.
Biswas N. 2017. Modified mesoporous silica nanoparticles for enhancing oral bioavailability and

t
antihypertensive activity of poorly water soluble valsartan. European Journal of Pharmaceutical

ip
Sciences. 99:152-160.
Chang D, Gao Y, Wang L, Liu G, Chen Y, Wang T, Tao W, Mei L, Huang L, Zeng X. 2016.
Polydopamine-based surface modification of mesoporous silica nanoparticles as pH-sensitive drug

cr
delivery vehicles for cancer therapy. Journal of colloid and interface science. 463:279-287.
Chang F-P, Kuang L-Y, Huang C-A, Jane W-N, Hung Y, Yue-ie CH, Mou C-Y. 2013. A simple plant

us
gene delivery system using mesoporous silica nanoparticles as carriers. Journal of Materials Chemistry
B. 1(39):5279-5287.
Chen X, Yao X, Wang C, Chen L, Chen X. 2015. Mesoporous silica nanoparticles capped with

Microporous and Mesoporous Materials. 217:46-53.

an
fluorescence-conjugated cyclodextrin for pH-activated controlled drug delivery and imaging.

Daglar B, Ozgur E, Corman M, Uzun L, Demirel G. 2014. Polymeric nanocarriers for expected
nanomedicine: current challenges and future prospects. RSC Advances. 4(89):48639-48659.
M
De Milito A, Fais S. 2005. Tumor acidity, chemoresistance and proton pump inhibitors. Future oncology.
1(6):779-786.
De Oliveira Freitas LB, de Melo Corgosinho L, Faria JAQA, dos Santos VM, Resende JM, Leal AS,
Gomes DA, de Sousa EMB. 2017. Multifunctional mesoporous silica nanoparticles for cancer-
d

targeted, controlled drug delivery and imaging. Microporous and Mesoporous Materials. 242:271-283.
e

Ebrahimi-Gatkash M, Younesi H, Shahbazi A, Heidari A. 2017. Amino-functionalized mesoporous

MCM-41 silica as an efficient adsorbent for water treatment: batch and fixed-bed column adsorption
pt

of the nitrate anion. Applied Water Science. 7(4):1887-1901.

Escudero-Ortiz V, Pérez-Ruixo JJ, Valenzuela B. 2013. Development and validation of a high-
performance liquid chromatography ultraviolet method for lapatinib quantification in human plasma.
ce

Therapeutic drug monitoring. 35(6):796-802.

Esfandyari J, Shojaedin-Givi B, Hashemzadeh H, Mozafari-Nia M, Vaezi Z, Naderi-Manesh H. 2020.
Capture and detection of rare cancer cells in blood by intrinsic fluorescence of a novel functionalized
Ac

diatom. Photodiagnosis and Photodynamic Therapy.101753.

Fallah-Bagheri A, Saboury AA, Ma’mani L, Taghizadeh M, Khodarahmi R, Ranjbar S, Bohlooli M,
Shafiee A, Foroumadi A, Sheibani N et al. 2012. Effects of silica nanoparticle supported ionic liquid
as additive on thermal reversibility of human carbonic anhydrase II. International Journal of Biological
Macromolecules. 51(5):933-938.
Faraji AH, Wipf P. 2009. Nanoparticles in cellular drug delivery. Bioorganic & medicinal chemistry.
17(8):2950-2962.
Galateanu B, Hudita A, Negrei C, Ion RM, Costache M, Stan M, Nikitovic D, Hayes AW, Spandidos DA,
Tsatsakis AM. 2016. Impact of multicellular tumor spheroids as an in vivo‑ like tumor model on
anticancer drug response. International journal of oncology. 48(6):2295-2302.
Grimaldi N, Andrade F, Segovia N, Ferrer-Tasies L, Sala S, Veciana J, Ventosa N. 2016. Lipid-based
nanovesicles for nanomedicine. Chemical Society Reviews. 45(23):6520-6545.
Hartono SB, Hadisoewignyo L, Yang Y, Meka AK, Yu C. 2016. Amine functionalized cubic mesoporous
silica nanoparticles as an oral delivery system for curcumin bioavailability enhancement.
Nanotechnology. 27(50):505605.
Hashemi N, Vaezi Z, Khanmohammadi S, Sohi AN, Masoumi S, Hruschka V, Wolbank S, Redl H,
Presen DM, Naderi-Manesh H. 2020. A novel fluorescent hydroxyapatite based on iron quantum
cluster template to enhance osteogenic differentiation. Materials Science and Engineering: C.110775.
He Q, Zhang J, Shi J, Zhu Z, Zhang L, Bu W, Guo L, Chen Y. 2010. The effect of PEGylation of
mesoporous silica nanoparticles on nonspecific binding of serum proteins and cellular responses.
Biomaterials. 31(6):1085-1092.
Hobbs SK, Monsky WL, Yuan F, Roberts WG, Griffith L, Torchilin VP, Jain RK. 1998. Regulation of
transport pathways in tumor vessels: role of tumor type and microenvironment. Proceedings of the
National Academy of Sciences. 95(8):4607-4612.
Hosseini M, Vaezi Z, Ganjali MR, Faridbod F, Abkenar SD. 2011. Fluorescence “Turn-On” chemosensor

t
for the selective detection of beryllium. Spectrochimica Acta Part A: Molecular and Biomolecular

ip
Spectroscopy. 83(1):161-164.
Jaroniec C, Gilpin R, Jaroniec M. 1997. Adsorption and thermogravimetric studies of silica-based amide

cr
bonded phases. The Journal of Physical Chemistry B. 101(35):6861-6866.
Kim JM, Chang SM, Kong SM, Kim K-S, Kim J, Kim W-S. 2009. Control of hydroxyl group content in
silica particle synthesized by the sol-precipitation process. Ceramics International. 35(3):1015-1019.

us
Koch KM, Reddy NJ, Cohen RB, Lewis NL, Whitehead B, Mackay K, Stead A, Beelen AP, Lewis LD.
2009. Effects of food on the relative bioavailability of lapatinib in cancer patients. Journal of clinical
oncology. 27(8):1191-1196. an
Li Z, Zhang Y, Feng N. 2019. Mesoporous silica nanoparticles: synthesis, classification, drug loading,
pharmacokinetics, biocompatibility, and application in drug delivery. Expert opinion on drug delivery.
16(3):219-237.
Liang X-J, Chen C, Zhao Y, Wang PC. 2010. Circumventing tumor resistance to chemotherapy by
M
nanotechnology. Multi-Drug Resistance in Cancer.467-488.
Luchini A, D’Errico G, Leone S, Vaezi Z, Bortolotti A, Stella L, Vitiello G, Paduano L. 2018. Structural
organization of lipid-functionalized-Au nanoparticles. Colloids and Surfaces B: Biointerfaces. 168:2-
d

9.
Ma'mani L, Nikzad S, Kheiri-Manjili H, Al-Musawi S, Saeedi M, Askarlou S, Foroumadi A, Shafiee A.
e

2014. Curcumin-loaded guanidine functionalized PEGylated I3ad mesoporous silica nanoparticles

KIT-6: practical strategy for the breast cancer therapy. Eur J Med Chem. 83:646-654.
pt

Maleki A, Hamidi M. 2016. Dissolution enhancement of a model poorly water-soluble drug, atorvastatin,
with ordered mesoporous silica: comparison of MSF with SBA-15 as drug carriers. Expert opinion on
ce

drug delivery. 13(2):171-181.

Mehrdad-Vahdati B, Pourhashem S, Sedghi M, Vaezi Z, Shojaedin-Givi B, Rashidi A, Naderi-Manesh H.
2019. A novel aspect of functionalized graphene quantum dots in cytotoxicity studies. Toxicology in
Vitro. 61:104649.
Ac

Mei X, Chen D, Li N, Xu Q, Ge J, Li H, Yang B, Xu Y, Lu J. 2012. Facile preparation of coating

fluorescent hollow mesoporous silica nanoparticles with pH-sensitive amphiphilic diblock copolymer
for controlled drug release and cell imaging. Soft Matter. 8(19):5309-5316.
Meléndez-Ortiz HI, Mercado-Silva A, García-Cerda LA, Castruita G, Perera-Mercado YA. 2013.
Hydrothermal synthesis of mesoporous silica MCM-41 using commercial sodium silicate. Journal of
the Mexican Chemical Society. 57(2):73-79.
Mobasseri R, Karimi M, Tian L, Naderi-Manesh H, Ramakrishna S. 2017. Hydrophobic lapatinib
encapsulated dextran-chitosan nanoparticles using a toxic solvent free method: fabrication, release
property & in vitro anti-cancer activity. Materials Science and Engineering: C. 74:413-421.
Mohammadi SS, Vaezi Z, Shojaedin-Givi B, Naderi-Manesh H. 2019. Chemiluminescent liposomes as a
theranostic carrier for detection of tumor cells under oxidative stress. Analytica chimica acta. 1059:
113-123
Muhammad F, Guo M, Qi W, Sun F, Wang A, Guo Y, Zhu G. 2011. pH-triggered controlled drug release
from mesoporous silica nanoparticles via intracelluar dissolution of ZnO nanolids. Journal of the
American Chemical Society. 133(23):8778-8781.
Muhammad F, Guo M, Wang A, Zhao J, Qi W, Guo Y, Zhu G. 2014. Responsive delivery of drug
cocktail via mesoporous silica nanolamps. Journal of colloid and interface science. 434:1-8.
Petrov KG, Zhang Y-M, Carter M, Cockerill GS, Dickerson S, Gauthier CA, Guo Y, Mook RA, Rusnak
DW, Walker AL. 2006. Optimization and SAR for dual ErbB-1/ErbB-2 tyrosine kinase inhibition in
the 6-furanylquinazoline series. Bioorganic & medicinal chemistry letters. 16(17):4686-4691.
Qi J, Qin B, Liu J, Yu Y, Zhang Z, Zhang W, Cai Q, Zhu W. 2011. MCM-41 single crystal of hexagonal
circular bicone with pseudo-singular surface and morphogenesis. CrystEngComm. 13(14):4666-4675.
Ravar F, Saadat E, Kelishadi PD, Dorkoosh FA. 2016. Liposomal formulation for co-delivery of
paclitaxel and lapatinib, preparation, characterization and optimization. Journal of liposome research.
26(3):175-187.

t
Rezaei N, Mehrnejad F, Vaezi Z, Sedghi M, Asghari SM, Naderi-Manesh H. 2020. Encapsulation of an

ip
endostatin peptide in liposomes: Stability, release, and cytotoxicity study. Colloids and Surfaces B:
Biointerfaces. 185:110552.

cr
Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, Cottin V, Flaherty KR, Hansell
DM, Inoue Y. 2014. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. New England
Journal of Medicine. 370(22):2071-2082.

us
Roy V, Perez EA. 2009. Beyond trastuzumab: small molecule tyrosine kinase inhibitors in HER-2–
positive breast cancer. The Oncologist. 14(11):1061-1069.
Sakhtianchi R, Darvishi B, Mirzaie Z, Dorkoosh F, Shanehsazzadeh S, Dinarvand R. 2019. Pegylated
an
magnetic mesoporous silica nanoparticles decorated with AS1411 Aptamer as a targeting delivery
system for cytotoxic agents. Pharmaceutical development and technology. 24(9):1063-1075.
Shah P, Rajput SJ. 2019. Investigation of in vitro permeability and in vivo pharmacokinetic behavior of
bare and functionalized MCM-41 and MCM-48 mesoporous silica nanoparticles: a burst and
M
controlled drug release system for raloxifene. Drug development and industrial pharmacy. 45(4):587-
602.
Siegel RA, Rathbone MJ. 2012. Overview of controlled release mechanisms. Fundamentals and
d

Applications of Controlled Release Drug Delivery. Springer; p. 19-43.

Tang F, Li L, Chen D. 2012. Mesoporous silica nanoparticles: synthesis, biocompatibility and drug
e

delivery. Advanced Materials. 24(12):1504-1534.

Vaezi Z, Sedghi M, Ghorbani M, Shojaeilangari S, Allahverdi A, Naderi-Manesh H. 2020. Investigation
pt

of the programmed cell death by encapsulated cytoskeleton drug liposomes using a microfluidic
platform. Microfluidics and Nanofluidics. 24(48): 48.
ce

Xia Y, Shi C-Y, Fang J-G, Wang W-Q. 2018. Approaches to developing fast release pellets via wet
extrusion-spheronization. Pharmaceutical development and technology. 23(5):432-441.
Yoncheva K, Popova M, Szegedi A, Mihály J, Tzankov B, Lambov N, Konstantinov S, Tzankova V,
Pessina F, Valoti M. 2014. Functionalized mesoporous silica nanoparticles for oral delivery of
Ac

budesonide. Journal of Solid State Chemistry. 211:154-161.

Yu Q, Qiu Y, Chen X, Wang X, Mei L, Wu H, Liu K, Liu Y, Li M, Zhang Z. 2019. Chemotherapy
priming of the Pancreatic Tumor Microenvironment Promotes Delivery and Anti-Metastasis Efficacy
of Intravenous Low-Molecular-Weight Heparin-Coated Lipid-siRNA Complex. Theranostics.
9(2):355.
Zeng X, Chen H, Zheng Y, Tao W, Fan Y, Huang L, Mei L. 2012. Enhanced adsorption of puerarin onto
a novel hydrophilic and polar modified post-crosslinked resin from aqueous solution. Journal of
colloid and interface science. 385(1):166-173.
Zhang D, Pal A, Bornmann WG, Yamasaki F, Esteva FJ, Hortobagyi GN, Bartholomeusz C, Ueno NT.
2008. Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast
cancer cells. Molecular cancer therapeutics. 7(7):1846-1850.
Zhang X, Zeng X, Liang X, Yang Y, Li X, Chen H, Huang L, Mei L, Feng S-S. 2014. The
chemotherapeutic potential of PEG-b-PLGA copolymer micelles that combine chloroquine as
autophagy inhibitor and docetaxel as an anti-cancer drug. Biomaterials. 35(33):9144-9154.
Zhang Y, Wang J, Bai X, Jiang T, Zhang Q, Wang S. 2012. Mesoporous silica nanoparticles for
increasing the oral bioavailability and permeation of poorly water soluble drugs. Molecular
pharmaceutics. 9(3):505-513.

Figure legends:

t
Scheme 1. Schematic presentation of the preparation of modified mesoporous nano-carrier and

ip
drug loading

cr
Figure 1. (a) XRD patterns, (b) SEM images, and (c) DLS size distribution of MCM-41 silica
NPs

us
Figure 2. FTIR spectra of (blue) ImIL-PEG@MCM-41, (pink) Lap, and (green) Lap@ImIL-
PEG@MCM-41 an
Figure 3. (a) N2 adsorption-desorption isotherms, and (b) pore size diameters from BJH of NPs
Table 1. N2 adsorption-desorption isotherms results of MCM-41, ImIL-PEG@MCM-41, and
M
Lap@ImIL-PEG@MCM-41
Figure 4. TGA curves of MCM-41, ImIL-PEG@MCM-41, and Lap@ImIL-PEG@MCM-41.
d

Figure 5. (a) Entrapment and (b) loading efficiency of Lap@ImIL-PEG@MCM-41 in correlation

e

with Lap/NP ratio

pt

Figure 6. Drug release pattern at pH 5 and pH 7.4

Figure 7. (A) Cytotoxicity graphs of (a) SKBR3 cell and (b) HEK-293 incubated with ImIL-
ce

PEG@MCM-41 (violet hatched), Free Lap (blue) and Lap@ImIL-PEG@MCM-41

(green dotted) for different incubation time (24 h and 48 h). (B) The statistical analysis
Ac

of IC50 for both cell lines, more statistical results are reported in ESM.
Figure 8. Fluorescent microscopy of SKBR3 cells (a) Control without treatment and were treated
with (b) free Lapatinib, (c) ImIL-PEG@MCM-41 (nano-DDS), (d) Lap@ImIL-
PEG@MCM-41 after 24 hours, and (e, f) fluorescence of FITC in the (c and d), cell
nucleus was stained by Hoechst 33342 with blue fluorescence; scale bar represents
100 μm
Figure 9. Fluorescent microscopic images of SKBR3 microspheres (i) bright field images of the
tumors, (ii) fluorescent images of the Lap@ImIL-PEG@MCM-41 and (iii) merged
images. After 12 hours of incubation with Lap@ImIL-PEG@MCM-41, the images has
taken every 12 hours and the control was not treated; scale bar represents 200 μm.

t
ip
cr
us
an
M
d

Scheme 1.
e
pt
ce
Ac
Ac
ce
pt
ed
Figure 1
M
an
us
cr
ip
t
Ac
ce
pt
ed
Figure 2
M
an
us
cr
ip
t
Ac
ce
pt
ed Figure 3
M
an
us
cr
ip
t
Ac
ce
pt
ed
Figure 4
M
an
us
cr
ip
t
Ac
ce
pt
ed Figure 5
M
an
us
cr
ip
t
Ac
ce
pt
ed
Figure 6
M
an
us
cr
ip
t
 t
ip
cr
us
an
M
e d

(B)
pt

IC50 Values (µg.mL-1)

Free Lap. ImIL-PEG@MCM-41 Lap@ImIL-PEG@MCM-41

ce

SKBR3- 24h 4.717 29.144 8.513

Ac

SKBR3- 48h 4.587 24.512 4.868

HEK293- 24h 10.026 N.A. 11.537

HEK293- 48h 9.203 N.A. 10.294

Figure 7
 Ac
ce
pt
ed

Figure 8
M
an
us
cr
ip
t
 Ac
ce
pt
ed

Figure 9
M
an
us
cr
ip
t
 Table 1

BET Pore size Pore volume BJH average

Compound Surface area pore diameter
(m2.g-1) (nm) (cm3.g-1) (nm)

MCM-41 689 3.62 0.789 1.71

ImIL-PEG@MCM-41 471 3.01 0.474 1.56

Lap@ImIL-PEG@MCM-41 362 2.50 0.163 1.27

t
ip
cr
us
Highlight


an
Imidazolium-based ionic liquid functionalized PEGylated mesoporous silica
nanoparticles MCM-41 was prepared
 High entrapment efficiency of [ImIL-PEG@MCM-41] was evaluated in Lapatinib
M
delivery to breast cancer cells
 It showed pH-responsive controlled and highly programmed release in vitro breast
cancer therapy
d

 The pure nanoparticles have no cytotoxicity against SKBR3, and HEK-293

e

 Biocompatibility, high loading and controllability make it a robust tool in

pt

biomedicine usage
ce
Ac
Graphical Abstract

t
ip
cr
The application of the modified mesoporous silica nanoparticles by ionic liquid for increase
loading of cationic Lapatinib drug

us
an
M
e d
pt
ce
Ac